Department of Chemistry and Chemical Biology, TU Dortmund University, 44227, Dortmund, Germany.
Max Planck Institute of Biochemistry, 82152, Martinsried, Germany.
Commun Biol. 2022 Mar 25;5(1):264. doi: 10.1038/s42003-022-03178-2.
The RNA phage MS2 is one of the most important model organisms in molecular biology and virology. Despite its comprehensive characterisation, the composition of the RNA replication machinery remained obscure. Here, we characterised host proteins required to reconstitute the functional replicase in vitro. By combining a purified replicase sub-complex with elements of an in vitro translation system, we confirmed that the three host factors, EF-Ts, EF-Tu, and ribosomal protein S1, are part of the active replicase holocomplex. Furthermore, we found that the translation initiation factors IF1 and IF3 modulate replicase activity. While IF3 directly competes with the replicase for template binding, IF1 appears to act as an RNA chaperone that facilitates polymerase readthrough. Finally, we demonstrate in vitro formation of RNAs containing minimal motifs required for amplification. Our work sheds light on the MS2 replication machinery and provides a new promising platform for cell-free evolution.
MS2 噬菌体是分子生物学和病毒学中最重要的模式生物之一。尽管它已经得到了全面的描述，但 RNA 复制机制的组成仍然不清楚。在这里，我们对重新构建体外功能性复制酶所需的宿主蛋白进行了表征。通过将纯化的复制酶亚复合物与体外翻译系统的元件结合，我们证实了三个宿主因子 EF-Ts、EF-Tu 和核糖体蛋白 S1 是活性复制酶全酶复合物的一部分。此外，我们发现翻译起始因子 IF1 和 IF3 调节复制酶的活性。虽然 IF3 直接与复制酶竞争模板结合，但 IF1 似乎作为 RNA 伴侣，促进聚合酶通读。最后，我们在体外形成了含有最小扩增所必需的基序的 RNA。我们的工作阐明了 MS2 复制机制，并为无细胞进化提供了一个新的有前途的平台。
